1. Field of the Invention
The present invention relates to a recombinant plasmid, a method of producing a transgenic fish, a host cell and transgenic animal containing it.
2. Description of the Prior Art
Transgenic fish studies make use of genes that are driven by both heterologous and homologous sources of regulatory element, and originate from constitutive or tissue-specific expression genes. Control elements include genes from antifreeze protein, mouse metallothionein, chicken δ-crystalline, carp β-actin, salmon histone H3 and carp α-globin and so on. However, there are important drawbacks to the use of these DNA elements in transgenic fish, including low expression efficiency and the mosaic expression of transgene patterns.
The microinjection into mekada eggs of lac reporter gene driven by the mekada β-actin promoter results in the transient expression of the lacZ gene, even in the F1 generation, though expression is low and highly mosaic. Hamada et al. reported a similar result in medaka embryos derived from eggs microinjected with green fluorescence protein fused with the medaka β-actin promoter (Hamada et al., 1998, Mol Marine Biol Biotechnol 7: 173-180).
Chi-Yuan Chou et al. disclosed a DNA construct flanked at both ends by ITRs to increase the efficient expression of transgenic genes in medaka. A uniform transgene expression was achieved in the F0 and the following two generations (Chi-Yuan Chou et al., 2001, Transgenic Research 10: 303-315). Moreover, Chung-Der Hsiao et al. indicated that the incorporation of AAV-ITPs into transgenes results in uniform gene expression in the F0 generation and stable transmission of transgenes in zebrafish (Chung-Der Hsiao et al., 2001, Developmental Dynamics 220:323-336).
The zebrafish, Danio rerio, is a new model organism for vertebrate developmental biology. As an experimental model, the zebrafish offers several major advantages such as easy availability of eggs and embryos, tissue clarity throughout embryogenesis, external development, short generation time and easy maintenance of both the adult and the young.
The known fluorescent genes such as GFP gene (including EGFP gene) have been introduced into zebrafish by using various gene promoters, including rat myosin light-chain enhancer (Moss, J. B. et al., Green fluorescent protein marks skeletal muscle in murine cell lines and zebrafish. Gene 173, 8998, 1996), zebrafish and tilapia insulin-like growth factor I promoter (Chen, J. Y et al., Isolation and characterization of tilapia (Oreochromis mossambicus) insulin-like growth factors gene and proximal promoter region (DNA Cell Biol. 17,359-376, 1998). All of these transgenic experiments aim at either developing a GFP transgenic system for gene expression analysis or at testing regulatory DNA elements in gene promoters.
WO0049150 discloses a fluorescent transgenic ornamental fish expressed by single fluorescent gene such as GFP. However, no fishes expressing two or more fluorescent genes uniformly and stably are developed.